A single N-linked glycosylation site is implicated in the regulation of ligand recognition by the I-type lectins CD22 and CD33.

CD22 is an immunoglobulin superfamily B lymphocyte-specific adhesion receptor and a member of the recently identified I-type class of lectins. Recent work has shown that CD22 specifically recognizes sialic acid linked alpha2,6 to terminal N-linked oligosaccharides on selected cell surface glycoproteins. CD22-ligand interaction is regulated by the activity of a beta-galactoside alpha2, 6-sialyltransferase that can inactivate CD22-mediated binding by sialylating the CD22 receptor itself. These observations suggest that N-linked glycosylation sites on the CD22 molecule may play a role in the regulation of CD22-mediated adhesion. In this work we have performed site-specific mutagenesis of potential N-linked glycosylation sites on CD22 in an effort to determine whether they might be involved in ligand recognition. We show that mutation of a single potential N-linked glycosylation site in the first immunoglobulin domain of CD22 completely abrogates ligand recognition. Interestingly, this site is characterized by the sequence NCT, where the cysteine is thought to be involved in an intrachain disulfide bond. Site-directed mutagenesis of similar NC(T/S) motifs in the first or second Ig domains of the I-type lectins myelin-associated glycoprotein, and sialoadhesin did not disrupt their ability to mediate sialic acid binding. In contrast, mutation of a NCS motif in the first Ig domain of the I-type lectin CD33 unmasked its sialic acid binding activity. These observations suggest that a single N-linked glycosylation site located at a similar position in the CD22 and CD33 glycoproteins is critical for regulating ligand recognition by both receptors.